PUMA mRNA as an indicator for islet quality assessment for transplantation

K. Omori¹, K. Ishiyama¹, G. Agrawal¹, I. Nair¹, J. Rawson¹, M. Mitsuhashi², I. Todorov¹, Y. Mullen^1
1 Southern California Islet Cell Resource Center, Beckman Research Institute of the City of Hope , Department of Diabetes, Endocrinology and Metabolism, Duarte, USA; ² Hitachi Chemical Research Center, Irvine, USA

Objective:Proinflammatory cytokines, including TNF-α, IL-1β and IFN-γ, are released during pancreas preservation, islet isolation and post-transplantation, inducing beta-cell death. A proapoptotic gene of the Bcl-2 family, PUMA (p53 up-regulated modulator of apoptosis), is involved in cytokine-mediated apoptosis through Nuclear Factor-KappaB (NF-κB) activation. Our aim was to examine the involvement of PUMA on beta-cell dysfunction/death through these processes.

Method:INS-1 cells and rat islets transfected with Puma siRNA were cold preserved for 18 hours. After returning to 37°C, the cells were exposed to IL-1β and IFN-γ for 8 or 48 hours. Beta-cell death was quantitated by FACS or Laser Scanning Cytometry. Puma protein expression was examined by western blot. RT-PCR was used to measure the expression of PUMA mRNA or preproinsulin (INS) pre-mRNA to assess beta-cell insulin synthesis. A marginal number of human islets or Puma siRNA transfected rat islets were also transplanted into STZ-diabetic NODscid mice.

Results:Puma protein and phosphorylated p65 component of NF-κB were up-regulated in rat islets during cold preservation. The cytokine stimulation significantly increased Puma expression and cell death in INS-1 cells, which was prevented by Puma siRNA transfection (p<0.03, n=3). In vitro, glucose-mediated insulin synthesis in human islets inversely correlated with cytokine mediated PUMA mRNA expression levels. In vivo, diabetes reversal in NODscid mice was significantly improved with human islets expressing lower levels of PUMA mRNA prior to transplantation (p<0.001, r=0.64, 24 mice receiving 10 different islet lots). Islets expressing lower PUMA mRNA also contained fewer apoptotic beta-cells. Silencing Puma transfecting with Puma siRNA improved rat islet function post-transplantation in diabetic NODscid mice.

Conclusions:Our results indicate that PUMA is involved in beta-cell death during cold preservation, islet isolation and after transplantation. PUMA mRNA and glucose induced INS pre-mRNA expression in human islets may be used to predict beta-cell function following transplantation.